Project summary For simplicity we tend to imagine that most proteins have a single mechanism of action. The retinoblastoma tumor suppressor is an example of a protein that defies this simple classification. RB1 is functionally inactivated in most human cancers and the molecular properties of its protein product (RB) have been studied intensively. Despite this research, RB's mechanism of action has remained an enigma. RB has been reported to physically associate with hundreds of proteins and many different interactions have been proposed to contribute to its tumor suppressive properties. The RB research community is faced with a conundrum: which of these interactions are real, which are not, and how could one protein co-ordinate its effects on so many potential targets? Recent studies from several laboratories have suggested that the answers to these questions lie in a code of RB phosphorylation. The concept is that normal cells do not contain a single form of RB, but that differential phosphorylation generates multiple isoforms of RB that have different binding properties and, presumably, perform different roles. In essence, the action of RB is tailored by phosphorylation. RB is known to have 14 sites of CDK phosphorylation. We have recently developed methods that allow us to use mass spectrometry-based proteomics to profile RB complexes. We have also generated panels of isogenic cell cultures in which we can replace the endogenous RB protein with mutant RB proteins that contain just a single cdk phosphorylation site, or a single phospho-mimicking mutation. In this application we propose to use these tools to decipher this code of RB phosphorylation. In Aim 1 we will use state-of-the-art proteomics to define the binding properties of each of the mono-phosphorylated isoforms of RB. In Aim 2 we will identify the functional consequences of these interactions by identifying the transcriptional programs that they control and by genomic loci that they target. Using this binding information and transcription profiles we will identify the molecular interactions that allow specific mono-phosphorylated isoforms of RB to control distinct programs of transcription. Together these experiments will generate a framework of molecular information that is critical to be able to understand RB's mechanism of action.